Search Results (30)

This case report describes the following scenario: A 56-day-old Arabian filly presented with classical symptoms of respiratory distress and a rapidly deteriorating condition, despite intensive antimicrobial treatment, resulting in death. The post-mortem examination revealed severe bronchopneumonia with characteristic disseminated pus-containing nodules. Microbiological tests and PCR confirmed the presence of R. equi carrying the virulence-associated protein A (VapA) gene. The isolate was sensitive to macrolides and fluoroquinolones but showed resistance or intermediate susceptibility to several commonly used antimicrobials. This case highlights the diagnostic and therapeutic challenges posed by this intracellular pathogen and emphasizes the importance of early detection, targeted therapy, and biosecurity measures—especially in the absence of an effective commercial vaccine. Full article

Despite the increased reporting of Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKp) in Egypt, there is a paucity of information regarding the molecular characteristics of such strains. Herein, we present the genome sequence of two CR-hvKp strains, K22 and K45, which were isolated from VAP (ventilator-associated-pneumonia) patients admitted to pediatric ICU at Assiut University Children’s Hospital, Egypt. K22 and K45 isolates were subjected to antimicrobial susceptibility testing and whole-genome sequencing. Genomic analysis was performed to characterize each strain, determining their plasmids, antimicrobial resistance (AMR) genes, and virulence determinants. K22 possessed an extensive drug resistance phenotype (XDR), whilst K45 exhibited a multidrug resistance phenotype (MDR), with genome sequencing revealing the presence of a diverse array of AMR genes. Both strains were resistant to the carbapenem antibiotic imipenem, carrying the OXA-48 carbapenemase, with K22 additionally possessing an NDM-1 carbapenemase. Each strain was considered high-risk, with K22 and K45 respectively belonging to sequence types ST383 and ST14 and possessing virulence genes implicated in hypervirulence (e.g., iucABCD-iutA and rmpA). Importantly, both strains carried multiple plasmid replicons, including an AMR/virulence IncHI1B/FIB hybrid plasmid and MDR IncL/M plasmids. This report highlights the critical role of plasmids in the evolution of virulent K. pneumoniae strains and suggests the circulation of an IncHI1B/FIB hybrid plasmid, simultaneously disseminating AMR and hypervirulence, amongst K. pneumoniae strains within Assiut University Children’s Hospital. Full article

Aeromonas salmonicida is a predominant pathogen that infects fish. The pathogen A. salmonicida subsp. masoucida (ASM) was isolated for the first time from diseased starry flounders (Platichthys stellatus). Our study aimed to isolate, characterize, and investigate the pathogenicity of ASM. Bacterial species were identified using 16s rRNA, gyrB, dnaJ, and vapA analyses. Phylogenetic tree analysis revealed that the ASM strains were clustered with the ASM ATCC strain and other strains isolated from black rockfish. In the antimicrobial susceptibility test, the three ASM strains were considered non-wild types for enrofloxacin, florfenicol, flumequine, oxolinic acid, and oxytetracycline susceptibility. Histopathological analysis revealed bacterial colonies in the secondary lamella and heart, indicating that ASM strains are highly virulent in fish. Comparative analysis and annotation via genome sequencing revealed that, among the 1156 factors, adherence factors were the most prevalent putative virulence determinants, followed by the effector delivery system and adherence. ASM was found to possess 43 type III secretion systems, 22 type VI secretion systems, 11 antimicrobial resistance genes, 3 stress genes, and prophage regions. These findings provide new insights into the virulence profile of ASM and highlight the risk posed by emerging pathogenic strains to starry flounders. Full article

Introduction: Nosocomial lower respiratory tract infections (nLRTIs), including hospital-acquired pneumonia (HAP) and ventilator-associated pneumonia (VAP), remain significant challenges due to high mortality, morbidity, and healthcare costs. Implementing accurate and timely diagnostic strategies is pivotal for guiding optimized antimicrobial therapy and addressing the growing threat of antimicrobial resistance. Areas Covered: This review examines emerging microbiological diagnostic methods for nLRTIs. Although widely utilized, traditional culture-based techniques are hindered by prolonged processing times, limiting their clinical utility in timely decision-making. Advanced molecular tools, such as real-time PCR and multiplex PCR, allow rapid pathogen identification but are constrained by predefined panels. Metagenomic next-generation sequencing (mNGS) provides comprehensive pathogen detection and resistance profiling yet faces cost, complexity, and interpretation challenges. Non-invasive methods, including exhaled breath analysis using electronic nose (e-nose) technology, gene expression profiling, and biomarker detection, hold promise for rapid and bedside diagnostics but require further validation to establish clinical applicability. Expert Opinion: Integrating molecular, metagenomic, biomarker-associated, and traditional diagnostics is essential for overcoming limitations. Continued technological refinements and cost reductions will enable broader clinical implementation. These innovations promise to enhance diagnostic accuracy, facilitate targeted therapy, and improve patient outcomes while contributing to global efforts to mitigate antimicrobial resistance. Full article

This study investigates the impact of nutritional supplementation on semen quality, epigenetic-related gene expression, and oxidative status in boars. Thirty boars were divided into a control group and a treatment group receiving Espermaplus (a supplement containing various vitamins, amino acids, omega-3 fatty acids, and trace elements with antioxidant properties). The experiment was performed for 12 weeks. Semen samples were collected at four moments: before starting the supplementation and after 3 weeks, 8 weeks, and 12 weeks. Spermatozoa concentration, motility, and kinematics were assessed using the CASA system. The measured parameters included curvilinear velocity—VCL; straight-line velocity—VSL; average path velocity—VAP; curvilinear distance—DCL; straight line distance—DSL; distance of average path—DAP; amplitude of lateral head displacement—ALH; beat-cross frequency—BCF; and head activity—HAC. Moreover, superoxide dismutase (SOD) and glutathione peroxidase (GPx) activity in seminal plasma, as well as the concentration of thiobarbituric acid reactive substances (TBARS), were measured to assess oxidative stress levels in boar’s seminal plasma. The expression of epigenetic-related genes such as Protamine 1 (Prm1), Protamine 2 (Prm2), and DNA-methyltransferase 3 alpha (Dnmt3a) were evaluated using real-time PCR. The treated group showed a significant increase in spermatozoa concentration (p = 0.003), total motility (p = 0.001), and progressive motility (p = 0.002) after 12 weeks compared to the control group. Kinematic parameters such as VCL, VSL, and VAP were also significantly higher (p < 0.001; p = 0.028; p < 0.001, respectively) in the treated group by the end of the experiment. SOD and GPx activities were consistently higher (p < 0.01; p < 0.001, respectively) in the treated group, indicating enhanced antioxidative capacity. TBARS levels as an indicator of lipid peroxidation and oxidative damage were significantly lower (p < 0.01) in the treated group by the end of the study. Significant changes were observed in the expression of epigenetic-related genes. The supplementation of boar diets with Espermaplus significantly improved semen quality, reduced oxidative stress, and had an impact on the expression levels of certain epigenetic-related genes, suggesting that dietary antioxidants and bioactive compounds can enhance boar semen. Full article

The pine wood nematode (Bursaphelenchus xylophilus), the pathogen of pine wilt disease (PWD), has caused enormous economic losses in Asian forests. Whether venom allergen proteins (VAPs) are involved in the accumulation of key defense substances in pine trees during the interaction between B. xylophilus and host trees, and their specific function as putative effectors secreted through stylets, has not been fully elucidated. In this study, the role of the BxVAP2 effector protein in the infection process was analyzed through bioinformatics and phylogenetic tree construction. The expression profile of BxVAP2 during infection was analyzed using qRT-PCR, and its expression under the stress of Pinus massoniana metabolites was examined. Toxicity assays were conducted through the Agrobacterium transient expression of BxVAP2 in Nicotiana benthamiana, and its subcellular localization was investigated. The results showed that BxVAP2 contains a CAP domain and shares close evolutionary relationships with venom allergen proteins from related species, such as Bursaphelenchus mucronatus, Aphelenchoides besseyi, Aphelenchoides fujianensis, and Meloidogyne graminicola. BxVAP2 was upregulated during the infection of P. massoniana, indicating that BxVAP2 is a key effector in the infection and colonization process of B. xylophilus and may play an important role during the rapid population growth phase. BxVAP2 responds to P. massoniana metabolites, where different concentrations of α-pinene suppressed its expression, while high concentrations of β-pinene promoted its expression. Subcellular localization revealed that BxVAP2 localizes to the cell membrane and nucleus. The transient expression of BxVAP2 in N. benthamiana induced programmed cell death and regulated pattern-triggered immunity marker genes. These findings suggest that BxVAP2 plays an important role in the interaction between B. xylophilus and its host, responding to terpene stress and triggering plant defense. Full article

Biofilm formation of clinical isolates of Aeromonas salmonicida subspecies salmonicida was compared using scarce (minimal M9 and ABTG w/o amino acids) and enriched nutrient media (Tryptone Soya broth) at 8 °C, 16 °C, and 25 °C using direct enumeration of viable cells in biofilm (log CFU), crystal violet staining (ODc) of the formed biofilm biomass, and liquid–air border biofilm formation (pellicle test). Whole-genome sequencing (WGS) was performed with the usage of an Oxford nanopore system by Genomics and Transcriptomics Labor Düsseldorf (Heinrich-Heine-Universität Düsseldorf, Germany). A bioinformatic analysis was performed with the usage of Geneious Prime^® 2023.0.4 (Biomatters, Inc., Boston, MA, USA). All data were analyzed using Statistica software version 13.0, and changes in biofilm production in correlation to changes in the type of nutritional medium and temperature were compared between groups using a one-way ANOVA analysis and Tukey’s test. All isolates formed biofilms in minimal M9 at 8 °C and 16 °C, and nine isolates failed to form biofilms in minimal M9 at 25 °C. In an ABTG medium, all isolates produced biofilms at 8 °C; however, three isolates at 16 °C and seven isolates at 25 °C failed to form any biofilms. Significant biofilm formation was observed in TSB at all temperatures. Some strains that formed a good biofilm in solid–liquid interface did not have the ability to form a pellicle (liquid–air border biofilm), and vice versa. In all cases of nutritional medium and temperature changes, there were statistically significant differences in the intensity of biofilm production, especially in the detected number of viable cells inside biofilms (log CFU, p < 0.005). Multiple biofilm-production genes, including polar flagella (filM) LuxR family (transcriptional regulators) and VapA family of histidine-kinase-associated genes, were sequenced from all studied isolates. Genetic differences based on geographical origin were not observed among the isolates. Significant variations in isolates’ ability to form biofilms were observed, possibly due to epigenetic factors. The optimal temperature for biofilm formation of A. salmonicida subspecies salmonicida in scarce media was 8 °C, and the majority of isolates were not capable of biofilm formation at 25 °C without enriched nutrient media. Full article

ProAKAP4 is a sperm structural protein that regulates motility through the PKA-dependent cAMP signaling pathway, which is synthesized as an X chromosome-linked member of the gene family. This study aims to determine the optimal level of proAKAP4 for evaluating sexed semen through investigating its relationship with the longevity of sperm quality in sexed Holstein bull sperm. A total of 30 sexed sperm samples (bearing X chromosomes) from 30 distinct Holstein bulls (n = 30) were analyzed. The frozen bull sperm samples were assessed for their proAKAP4 levels, mitochondrial membrane potential, plasma membrane and acrosome integrity (PMAI), and spermatozoa movement parameters at hours 0 and 3 after thawing. The proAKAP4 levels in the sexed sperm samples ranged from 16.35 to 72.10 ng/10 M spz, with an average of 37.18 ± 15.1 ng/10 M spz. A strong positive correlation was observed between proAKAP4 levels and total motility, progressive motility, PMAI, high mitochondrial membrane potential, VAP, and VCL values after 3 h of incubation, when compared to post-thaw analyses. The results also reveal that spermatozoa with proAKAP4 levels of ≥40 ng/10 M spz exhibit higher quality. In conclusion, the level of proAKAP4 in sexed sperm aligns with previous studies and shows potential as a biomarker for assessing the longevity of sexed sperm quality. Full article

Carbapenem-resistant Acinetobacter baumannii (CRAB) infections are associated with poor outcomes depending on patient’s conditions, clinical severity and type of infection, and treatment is challenging given the limited therapeutic options available. The aim of this study was to describe the clinical and microbiological characteristics of two outbreaks caused by CRAB in an intensive care unit (ICU). In addition, the mechanisms of resistance detected in these strains and the treatment chosen according to the available therapeutic options were analyzed. Overall, 28 patients were included. Ten patients (35.71%) had ventilator-associated pneumonia (VAP), ten (35.71%) had a bloodstream infection (BSI), and eight (28.57%) were only colonized. Recurrent infection occurred in 25% (5/20) of infected patients. Two different strains of A. baumannii were isolated from the index patient of the first outbreak. The first strain belonged to the ST85 and carried the bla_NDM-1 carbapenemase gene, while the second belonged to the ST2 and carried bla_OXA-23, and bla_OXA-66 carbapenemase genes. The phylogenetic analysis revealed that the ST2 strain was the cause of the major outbreak, and mutations in the AmpC gene were related to progressive increasing minimum inhibitory concentration (MIC) and finally, cefiderocol-resistance in one strain. The CRAB isolates from the second outbreak were also identified as ST2. Cefiderocol-resistant strains tests identified by the disc diffusion method were involved in 24% (6/25) of nosocomial infections. Using broth microdilution (BMD) ComASP^® only, 33.3% (2/6) of these strains were cefiderocol-resistant. All-cause ICU mortality was 21.4%. Conclusions: Cefiderocol is the first approved siderophore cephalosporin for the treatment of CRAB infections. Cefiderocol-resistant strains were related with bla_NDM-1 carbapenemase and mutations in the AmpC gene. Cefiderocol-resistant strains or that cannot be properly interpreted by disk diffusion, should be retested using BMD for definitive categorization. Full article

Bacterial ubiquitous Toxin-Antitoxin (TA) systems are considered to be important survival mechanisms during stress conditions. In regular environmental conditions, the antitoxin blocks the toxin, whereas during imbalanced conditions, the antitoxin concentration decreases, exposing the bacteria cell to a range of toxic events. The most evident consequence of this disequilibrium is cell growth arrest, which is the reason why TAs are generally described as active in the function of bacterial growth kinetics. Virulence-associated proteins B and C (VapBC) are a family of type II TA system, in which VapC is predicted to display the toxic ribonuclease activity while VapB counteracts this activity. Previously, using in silico data, we designated four VapBC TA modules in Leptospira interrogans serovar Copenhageni, the main etiological agent of human leptospirosis in Brazil. The present study aimed to obtain the proteins and functionally characterize the VapBC-1 module. The expression of the toxin gene vapC in E. coli did not decrease the cell growth rate in broth culture, as was expected to happen within active TA modules. However, interestingly, when the expression of the toxin was compared to that of the complexed toxin and antitoxin, cell viability was strongly affected, with a decrease of three orders of magnitude in colony forming unity (CFU). The assumption of the affinity between the toxin and the antitoxin was confirmed in vivo through the observation of their co-purification from cultivation of E. coli co-expressing vapB-vapC genes. RNAse activity assays showed that VapC-1 cleaves MS2 RNA and ribosomal RNA from L. interrogans. Our results indicate that the VapBC-1 module is a potentially functional TA system acting on targets that involve specific functions. It is very important to emphasize that the common attribution of the functionality of TA modules cannot be defined based merely on their ability to inhibit bacterial growth in a liquid medium. Full article

Antarctic krill (Euphausia superba) is a key species of the Antarctic ecosystem whose unique ecological status and great development potential have attracted extensive attention. However, the genomic characteristics and potential biological functions of the symbiotic microorganisms of Antarctic krill remain unknown. In this study, we cultured and identified a strain of Brucella sp. WY7 from Antarctic krill using whole-genome sequencing and assembly, functional annotation, and comparative genomics analysis. First, based on 16S rDNA sequence alignment and phylogenetic tree analysis, we identified strain WY7 as Brucella. The assembled genome of strain WY7 revealed that it has two chromosomes and a plasmid, with a total genome length of 4,698,850 bp and an average G + C content of 57.18%. The DNA—DNA hybridization value and average nucleotide identity value of strain WY7 and Brucella anthropi ATCC^® 49188^TM, a type strain isolated from human clinical specimens, were 94.8% and 99.07%, respectively, indicating that strain WY7 is closely related to Brucella anthropi. Genomic island prediction showed that the strain has 60 genomic islands, which may produce HigB and VapC toxins. AntiSMASH analysis results showed that strain WY7 might produce many secondary metabolites, such as terpenes, siderophores and ectoine. Moreover, the genome contains genes involved in the degradation of aromatic compounds, suggesting that strain WY7 can use aromatic compounds in its metabolism. Our work will help to understand the genomic characteristics and metabolic potential of bacterial strains isolated from Antarctic krill, thereby revealing their roles in Antarctic krill and marine ecosystems. Full article

This study aimed to explore the molecular epidemiology of Staphylococcus aureus isolated from patients on mechanical ventilation and the participation of virulence factors in the development of ventilator-associated pneumonia (VAP). A prospective cohort study was conducted on patients under mechanical ventilation, with periodic visits for the collection of tracheal aspirates and clinical data. The S. aureus isolates were analyzed regarding resistance profile, virulence, expression of protein A and alpha-toxin using Western blot, clonal profile using PFGE, sequence type using MLST, and characterization and quantification of phenol-soluble modulins. Among the 270 patients in the study, 51 S. aureus strains were isolated from 47 patients. The incidence density of S. aureus and MRSA VAP was 2.35/1000 and 1.96/1000 ventilator days, respectively; of these, 45% (n = 5) were resistant to oxacillin, with 100% (n = 5) harboring SCCmec types II and IV. The most frequent among the tested virulence factors were icaA, hla, and hld. The clonal profile showed a predominance of sequence types originating from the community. Risk factors for VAP were the presence of solid tumors and the sea gene. In conclusion, patient-related risk factors, together with microbiological factors, are involved in the development of S. aureus VAP, which is caused by the patient’s own strains. Full article

Mycobacterium bovis BCG is the only vaccine against tuberculosis. The variable forms of cultivation throughout the years, before seed-lots were developed, allowed in vitro evolution of the original strain, generating a family of vaccines with different phenotypic and genotypic characteristics. Molecular studies revealed regions of difference (RDs) in the genomes of the various BCG strains. This work aims to characterize the gene pair rv3407-rv3408 (vapB47-vapC47), coding for a toxin–antitoxin system of the VapBC family, and to evaluate possible transcriptional effects due to the adjacent BCG Moreau-specific genomic deletion RD16. We show that these genes are co-transcribed in BCG strains Moreau and Pasteur, and that the inactivation of an upstream transcriptional repressor (Rv3405c) due to RD16 has a polar effect, leading to increased vapBC47 expression. Furthermore, we detect VapB47 DNA binding in vitro, dependent on a 5′ vapB47 sequence that contributes to a palindrome, spanning the promoter and coding region. Our data shed light on the regulation of VapBC systems and on the impact of the BCG Moreau RD16 deletion in the expression of adjacent genes, contributing to a better understanding of BCG Moreau physiology. Full article

Genes of host plants and parasitic nematodes govern the plant–nematode interaction. The biological receptors and parasitism effectors are variable among plant species and nematode populations, respectively. In the present study, hatch testing and bioassays on cabbage, oilseed radish, and mustard were conducted to compare the biological characteristics among six populations of the beet cyst nematode Heterodera schachtii. Genetic patterns of the vap1 gene for the studied populations were distinct as shown by denaturing the gradient gel electrophoresis of PCR-amplified gene fragments. Concurrently, significant differences in the hatching rates, number of penetrated J2 in roots, and eggs/cyst ratios among the six nematode populations for the three cruciferous species were observed. In conclusion, analyzing the population genetic structure of H. schachtii plays a pivotal role in illustrating the variability in the plant–nematode interaction among its populations and plant species, which in its role leads to developing nematode management depending on plant resistance. Full article

During November to December 2020, a high rate of COVID-19-associated pneumonia with bacterial superinfections due to multidrug-resistant (MDR) pathogens was recorded in a COVID-19 hospital in Zagreb. This study analyzed the causative agents of bacterial superinfections among patients with serious forms of COVID-19. In total, 118 patients were hospitalized in the intensive care unit (ICU) of the COVID-19 hospital. Forty-six out of 118 patients (39%) developed serious bacterial infection (VAP or BSI or both) during their stay in ICU. The total mortality rate was 83/118 (70%). The mortality rate due to bacterial infection or a combination of ARDS with bacterial superinfection was 33% (40/118). Six patients had MDR organisms and 34 had XDR (extensively drug-resistant). The dominant species was Acinetobacter baumannii with all isolates (34) being carbapenem-resistant (CRAB) and positive for carbapenem-hydrolyzing oxacillinases (CHDL). One Escherichia coli causing pneumonia harboured the bla_CTX-M-15 gene. It appears that the dominant resistance determinants of causative agents depend on the local epidemiology in the particular COVID center. Acinetobacter baumannii seems to easily spread in overcrowded ICUs. Croatia belongs to the 15 countries in the world with the highest mortality rate among COVID-19 patients, which could be in part attributable to the high prevalence of bacterial infections in local ICUs. Full article